Metal ions and phosphatidylinositol 4,5-bisphosphate as interacting effectors of α-type plant phospholipase D

Abstract

Plant phospholipases D (PLD) are typically characterized by a C2 domain with at least two Ca²⁺ binding sites. In vitro, the predominantly expressed α-type PLDs need 20-100 mM CaCl₂ for optimum activity, whereas the essential activator of β- or γ-type PLDs, phosphatidylinositol 4,5-bisphosphate (PIP₂), plays a secondary role. In the present paper, we have studied the interplay between PIP₂ and metal ion activation of the well-known α-type PLD from cabbage (PLDα). With mixed micelles containing phosphatidyl-p-nitrophenol as substrate, PIP₂-concentrations in the nanomolar range are able to activate the enzyme in addition to the essential Ca²⁺ activation. Mg²⁺ ions are able to replace Ca²⁺ ions but they do not activate PLDα. Rather, they abolish the activation of the enzyme by Ca²⁺ ions in the absence, but not in the presence, of PIP₂. The presence of PIP₂ causes a shift in the pH optimum of PLDα activity to the acidic range. Employing fluorescence measurements and replacing Ca²⁺ by Tb³⁺ ions, confirmed the presence of two metal ion-binding sites, in which the one of lower affinity proved crucial for PLD activation. Moreover, we have generated a homology model of the C2 domain of this enzyme, which was used for Molecular Dynamics (MD) simulations and docking studies. As is common for C2 domains, it shows two antiparallel β-sheets consisting of four β-strands each and loop regions that harbor two Ca²⁺ binding sites. Based on the findings of the MD simulation, one of the bound Ca²⁺ ions is coordinated by five amino acid residues. The second Ca²⁺ ion induces a loop movement upon its binding to three amino acid residues. Docking studies with PIP₂ reveal, in addition to the previously postulated PIP₂-binding site in the middle of the β-sheet structure, another PIP₂-binding site near the two Ca²⁺ ions, which is in accordance with the experimental interplay of PIP₂, Ca²⁺ and Mg²⁺ ions.

Keywords: Activity; Brassica oleracea convar. capitata var. alba L.; Brassicaceae; C2 domain; Calcium; Fluorescence; Homology modelling; Mixed micelles; Molecular docking; PIP2; Phospholipase D.